Masaya Ueno. Masahiko Kobayashi. Naho Nomura. Ha Vu Thi. Hui Peng. Ahmed M. Hegazy. Masayoshi Masuko. Hirohito Sone. Fumio Arai. Atsushi Tajima.
Functional dissection of hematopoietic stem cell populations with a stemness-monitoring system based on NS-GFP transgene expression
Mohamed A.E. Ali Kyoko Fuse Yuko Tadokoro Takayuki Hoshii Masaya Ueno Masahiko Kobayashi Naho Nomura Ha Vu Thi Hui Peng Ahmed M. Hegazy Masayoshi Masuko Hirohito Sone Fumio Arai Atsushi Tajima Atsushi Hirao
Supplementary Figure 1. Analysis of the correlation between NS-GFP and endogenous NS mRNA. (a) Bone marrow mononuclear cells (MNCs) were fractionated into four populations according to NS-GFP intensity. (b) MNC GFP mRNA expression level. (c) MNC NS mRNA expression level. (d) Bone marrow LSK cells were fractionated into four populations according to NS-GFP intensity. (e) LSK GFP mRNA expression level. (f) LSK NS mRNA expression level. Data are presented as the mean ± SD of the ratios of GFP or NS to -actin mRNA copy numbers in fractionated cells (n = 3); ND, not detected.
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1Gy Irradiated CD48-LSK high
NS-GFP
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20%
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20%
20%
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Supplementary Figure 2. Repopulating capacity of HSCs after radiation. (a) FACS was used to separate 1 Gy–irradiated bone marrow CD48-LSK cells into five fractions according to NS-GFP intensity, of which three fractions—the top 20% (NS-GFPhigh), middle 20% (NSGFPmiddle), and bottom 20% (NS-GFPlow)—were used in subsequent experiments. 200 cells were transplanted per mouse. (b) Only the NS-GFPhigh fraction of cells shows long-term reconstitution capacity. Data shown are the mean frequencies of Ly5.2+ cells in the peripheral blood ± SD (n = 5). (c) Lineage analysis of transplanted fractions showing that only NSGFPhigh has multilineage chimerism 16 weeks after transplantation. Data shown are the mean frequencies of Ly5.2+ cells among B cells, T cells or myeloid cells ± SD (n = 5). ***p < 0.001; ND, not detected.
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NS-GFPhigh 0.77%
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Supplementary Figure 3. Repopulating capacity of cells in the LT-HSC and CD34+LSK compartments highly expressing NS-GFP. (a) FACS pattern of sorting LT-HSCs and CD34+ LSK cells with high levels of NS-GFP intensity. (b) The transplanted fractions show comparable total repopulation capacity in peripheral blood. Ten cells were transplanted per mouse. Data shown are the mean frequencies of Ly5.2+ cells in the peripheral blood ± SD (n = 5). (c) Lineage analysis of transplanted cells 16 weeks after transplantation showing comparable multilineage chimerism. Data shown are the mean frequencies of B cells, T cells or myeloid cells among Ly5.2+ cells ± SD (n = 5). Note: The data of "NS-GFPhigh (LT-HSC)" are identical to the "NS-GFPhigh of LT-HSCs" data presented in Figure 6. ns; not significant. B; B cells. M; myeloid cells. T; T cells.
